Detection of Xanthomonas oryzae pv oryzae from seeds and leaves of rice (Oryza sativa) using hrp gene based BIO-PCR marker

DINESH SINGH; SHWETA SINHA; RAVINDER PAL SINGH

doi:10.56093/ijas.v85i4.47932

Authors

DINESH SINGH Principal Scientist, Indian Agricultural Research Institute, New Delhi 110 012
SHWETA SINHA Former Senior Research Fellow, Division of Plant Pathology, Department of Biotechnology, Adhunik Institute of Education and Research, Ghaziabad, Uttar Pradesh 201 206
RAVINDER PAL SINGH Student, Department of Biotechnology, Adhunik Institute of Education and Research, Ghaziabad, Uttar Pradesh 201 206

https://doi.org/10.56093/ijas.v85i4.47932

Keywords:

BIO-PCR, Detection, hrp gene, Rice, Seed, Xanthomonas oryzae pv. oryzae

Abstract

Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae is a serious disease of rice. Detection of pathogen from infected plant parts and seeds is important to eliminate pathogen from disease cycle. For detection of X. oryzae pv. oryzae from natural infected leaves and stored seeds, a polymerase chain reaction technique using hrp gene (hypersensitive response and pathogenicity) sequence of X. oryzae pv. oryzae based primers DXoo_hrp1F (5’- GGACCCTCCGGTTCTGAG-3’) and DXoo_hrp1R (5’-CTGCAGGCATTGCTTAAAGA-3’) were used to amplify at 384 bp DNA fragment. The specificity of the primer was tested with X. oryzae pv. oryzae along with other Xanthomonas species such as X. campestris pv. campestris, X. axonopodis pv. punicae and Ralstonia solanacearum. It amplified only DNA fragment of X. oryzae pv. oryzae at 384 bp. The developed set of primer was highly sensitive and detected 2.6 × 102 cfu/ ml of bacteria. The X. oryzae pv. oryzae was detected from naturally infected leaves of rice collected from the bacterial ooze as DNA template through conventional – PCR. To detect X. oryzae pv. oryzae from seeds, the bacterial culture was artificially inoculated on rice cv. Pusa Basmati 1 plant at flowering time and the matured seeds were harvested and stored for 8 months under ambient conditions. The bacterium was detected from seeds after 8 months of storage even 2 seeds/ml, when DNA of bacterium was extracted by short cut methods adding 95% ethanol through BIO-PCR. This method is reliable, more sensitive than conventional–PCR and may detect X. oryzae pv oryzae within 2 days from the seeds and other planting materials.

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References

Adachi N and Oku T. 2009. PCR – mediated detection of Xanthomonas oryzae pv. oryzae by amplification of the 16S- 23S r DNA spacer region sequence. Journal of General Plant Pathology 66: 303–9. DOI: https://doi.org/10.1007/PL00012969

Bassam B J, Cactano- Anolles G and Gresshoff P M. 1992. DNA amplification fingerprinting of bacteria. Applied Microbiology and Biotechnology 38: 70–6. DOI: https://doi.org/10.1007/BF00169422

Berg T, Tesoriero and Hailstones D L. 2005. PCR – based detection of Xanthomonas campestris pathovars in Brassica seed. Plant Pathology 54: 416–27. DOI: https://doi.org/10.1111/j.1365-3059.2005.01186.x

Di M, Ye H, Schaad N W and Roth D A. 1991. Selective recovery of Xanthomonas spp. from rice seed. Phytopathology 81: 1 358– 63. DOI: https://doi.org/10.1094/Phyto-81-1358

George M L C, Quinto V, Villamayor M and Nelson R J. 1996. A simple and rapid method for isolation of bacterial genomic DNA. International Rice Research Notes 21(2/3): 84.

Leit R P Jr, Minsavage G V, Bonas U and Stall R E. 1994. Detection and identification of phytopathogenic Xanthomonas strains by amplification of DNA sequences related to the hrp genes of Xanthomonas campestris pv vesicatoria. Applied and Environmental Microbiology 60: 1 068–77. DOI: https://doi.org/10.1128/aem.60.4.1068-1077.1994

Marefat A, Ophel Keller K, Scott E S and Sedgley M. 2006. The use of ARMS PCR in detection and identification of Xanthomonads associated with pistachio dieback in Australia. European Journal of Plant Pathology 116 (1): 57–68. DOI: https://doi.org/10.1007/s10658-006-9038-z

Mew T W, Alvarez A M, Leach J E and Swings J. 1993. Focus on bacterial blight of rice. Plant Disease 77: 5–12. DOI: https://doi.org/10.1094/PD-77-0005

Osdaghi E, Shams- bakhsh M, Alizadeh A and Lak M R. 2010. Study on common bean seed lots for contamination with Xanthomonas axonopodis pv. phaseoli BIO- PCR technique. International Journal of Agriculture 6 (3): 503–13.

Pitcher D G, Saunders N A and Owen R J. 1989. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Letters in Applied Microbiology 8: 151–6 DOI: https://doi.org/10.1111/j.1472-765X.1989.tb00262.x

Sakthivel N, Mortensen N C and Mathur S B. 2001. Detection of Xanthomonas oryzae pv. oryzae in artificially inoculated and naturally infected rice seeds and plants by molecular techniques. Applied Microbiology and Biotechnology 56: 435–41. DOI: https://doi.org/10.1007/s002530100641

Schaad N W, Cheong S S, Tamaki S, Hatziloukas E and Panopoulas N J. 1995. A combined biological and anzymatic amplification technique to detect Pseudomonas syringae pv. phaseolicola in bean seed extracts. Phytopathology 85: 243–8. DOI: https://doi.org/10.1094/Phyto-85-243

Schaad N W, Frederick R D, Shaw J, Schneider W L, Hickson R, Michael D P and Luster D G. 2003. Advances in molecularbased diagnostics in meeting crop biosecurity and phytosanitary issues. Annual Review of Phytopathology 41: 305–24. DOI: https://doi.org/10.1146/annurev.phyto.41.052002.095435

Schaad N W, Jones J B and Chun W. 2001. Laboratory Guide for the Identification of Plant Pathogenic Bacteria, 3rd Edn. American Phytopathological Society, St Paul, MN.

Singh D and Shri Dhar 2011. Bio-PCR based diagnosis of Xanthomonas campestris pathovars in black rot infected leaves of crucifers. Indian Phytopathology 64 (1): 7–11.

Valluvaparadedasan V and Mriappan V. 1989. Alternate hosts of rice bacterial blight (BB) pathogen Xanthomonas campestris pv. oryzae. International Rice Research Newsletter 14 (5): 27– 8.